The Identification and Characterization of a Noncontinuous Calmodulin-binding Site in Noninactivating Voltage-dependent KCNQ Potassium Channels
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m204130200
ISSN1083-351X
AutoresEva Yus-Nájera, Irene Santana-Castro, Álvaro Villarroel,
Tópico(s)Insect and Pesticide Research
ResumoWe show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two α-helices (A and B). These two helices encompass ∼85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of ∼130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1–5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels. We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two α-helices (A and B). These two helices encompass ∼85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of ∼130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1–5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels. calmodulin apo-calmodulin, Ca2+-free calmodulin Ca2+-bound calmodulin calmodulin binding motif glutathione S-transferase 5-dimethylaminonaphthalene-1-sulfonyl amino acid(s) The KCNQ transmembrane proteins are members of a family of voltage-dependent potassium selective channels that are involved in the control of cellular excitability. Remarkably, mutations in four of the five known members of this family have been associated with different hereditary human disorders. While mutations in the KCNQ1 subunit (KvQT1) lead to arrhythmia in the human long QT syndrome, mutations in KCNQ2 or KCNQ3 are associated with a benign form of epilepsy. It has also been shown that KCNQ4 is mutated in a dominant form of progressive hearing loss (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar).With regards to the normal physiology of this protein family, the KCNQ2 and KCNQ3 subunits have been shown to form M-type potassium channels whose expression is restricted to neuronal tissue (2Wang H.S. Pan Z. Shi W. Brown B.S. Wymore R.S. Cohen I.S. Dixon J.E. McKinnon D. Science. 1998; 282: 1890-1893Crossref PubMed Scopus (1015) Google Scholar). Moreover, in some brain areas and neuronal tissues, KCNQ4 and KCNQ5 also contribute to the formation of M channels, suggesting that the different combinations of KCNQ subunits may be in part responsible for the diversity of M channel properties (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar). The M current (I M) is a subthreshold noninactivating voltage-dependent potassium current that is found in many neuronal cell types. The M current controls membrane excitability, and it has been shown to be modulated by a variety of intracellular signals that in turn dramatically affect the firing rate of neurons. Among those intracellular signals, Ca2+ has been shown to mediate the inhibition of I M by B2bradykinin receptors in sympathetic neurons (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar). Indeed, intracellular Ca2+ can suppress the activity of M channels under conditions that do not support enzymatic activities such as phosphorylation (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This phenomenon suggests that an intermediary might be involved in this Ca2+-dependent modulation.In a search for candidates that might mediate the effects of Ca2+ in modulating I M, we screened a human brain cDNA library using the yeast two-hybrid system. We found that calmodulin (CaM)1bound to the C-terminal region of KCNQ channels. CaM is a small Ca2+-binding protein that acts as a ubiquitous intracellular Ca2+ sensor in the regulation of a growing diverse array of ion channels (5Saimi Y. Kung C. Annu. Rev. Physiol. 2002; 64: 289-311Crossref PubMed Scopus (290) Google Scholar). Efforts to define common characteristics of CaM binding have indicated that it associates with short α-helical sequences within its targets (6O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 7Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar, 8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar). However, it appears that the interaction of CaM with KCNQ channels does not conform to this simple model. Rather, our data suggest that the CaM-binding site in KCNQ channels is formed by two α-helices that are separated by a stretch of ∼130 amino acids. We hypothesize that those two helices come into close proximity in the tertiary structure, facilitating CaM binding.DISCUSSIONWe have demonstrated here that CaM binds to the intracellular C-terminal domain of neuronal KCNQ2, KCNQ3, and KCNQ5 transmembrane channels. Two-hybrid experiments suggest that CaM also binds to KCNQ1 and KCNQ4, but more experiments are necessary to unequivocally confirm this interaction. The voltage-dependent channels to which members of this family of proteins contribute have been implicated in a variety of physiological processes and pathologies. As a result, the modulation of the activity of these channels through intracellular signaling is important in maintaining the physiological homeostasis of nervous tissue (30Marrion N.V. Annu. Rev. Physiol. 1997; 59: 483-504Crossref PubMed Scopus (294) Google Scholar). An example of this can be seen in the Ca2+-dependent regulation of M channels (made up of KCNQ subunits) that influences the firing rate of the sympathetic cells in which they are expressed. Our results indicate that through its association with these proteins, CaM may mediate the Ca2+-dependent modulation of channels that include KCNQ subunits.In addition to demonstrating here that CaM associates with members of the KCNQ family, we have also defined its binding site. The binding site identified in this study is unusual in that it is composed of two discontinuous regions, helix A and helix B. Helix A contains an IQ-like binding motif, a motif (IQXXXRXXXXR) that mediates apo-CaM binding in a variety of proteins (31Bahler M. Rhoads A. FEBS Lett. 2002; 513: 107-113Crossref PubMed Scopus (361) Google Scholar) and that contains positively charged residues at positions 6 and 11. When compared with other IQ motifs, in the helix A of the KCNQ channels the second Arg is replaced by a negatively charged or neutral amino acid. As a result, this domain resembles the second "incomplete" IQ motif on myosin II, the region to which the regulatory myosin light chain (structurally similar to CaM) binds. A model of apo-CaM binding derived from this and other light chain structures bound to myosin IQ motifs reveals that the initial portion of this motif (IQXXXR) is the most critical part. Moreover, it is this region that is specifically recognized by the loop between EF hands 3 and 4 and that determines a semi-open lobe conformation (32Houdusse A. Silver M. Cohen C. Structure. 1996; 4: 1475-1490Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In accordance with this model, the interaction of KCNQ2 with CaM is destabilized when point mutations are introduced in this first part of the IQ motif.On many occasions, the C lobe of CaM has been shown to be that which interacts with peptides, as also occurs with CaM-like proteins that associate with peptides. Moreover, the bound peptide essentially occupies the same position relative to the C-terminal EF hand domain (33Atkinson R.A. Joseph C. Kelly G. Muskett F.W. Frenkiel T.A. Nietlispach D. Pastore A. Nat. Struct. Biol. 2001; 8: 853-857Crossref PubMed Scopus (73) Google Scholar). The finding that the C lobe is sufficient to bind to SK channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar), P/Q Ca2+ channels, and neurogranin supports this finding. However, in contrast, it appears that to bind to KCNQ2 or KCNQ3 channels, both the C and N lobes are required.Surprisingly, we found that when using the full-length C-terminal region of SK2 as bait, point mutations that abolish Ca2+binding to EF hands 3 or 4 also abrogate the interaction with CaM. There is, however, ample biochemical, functional, and structural evidence to indicate that Ca2+ is not bound to EF hands 3 or 4 when CaM interacts with SK2. In addition, it has been shown that the Ca2+-free C-terminal lobe is that which mediates the binding of CaM to the SK2 CaM-binding domain (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar). Our results indicate that mutating the first aspartate to alanine in the EF hands that directly mediate the interaction of apo-CaM with the target causes a reduction in the binding strength. By analogy with the SK2 CaM-binding domain, the observation that the interaction of CaM with KCNQ2 or KCNQ3 does not tolerate point mutations at EF hand 1 and (to a lesser degree) 3 suggests that binding to the KCNQ-BD is mainly mediated by the apo-EF hands 1 and 3. Interestingly, EF hand 1 is most similar to EF hand 3 (8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar), suggesting that both play a similar role in stabilizing the target complex.The difficulties in identifying apo-CaM interactions have been highlighted by Erickson et al. (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). To overcome this problem, a very elegant technique has been developed, three-cube fluorescence resonance energy transfer, that demonstrates the preassociation of CaM with calcium channels in living cells (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). The yeast two-hybrid system is another viable alternative for approaching this problem. Our results provide further evidence that the two-hybrid assay is capable of detecting interactions between the Ca2+-free form of CaM and target proteins such as the SK2 K+ channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar). It should be born in mind that a limitation of the two-hybrid system is that the transmembrane segments of target proteins must be eliminated to allow targeting of the bait to the nucleus (34Niethammer M. Sheng M. Methods Enzymol. 1998; 293: 104-122Crossref PubMed Scopus (26) Google Scholar). However, the main advantage is that this is a relatively simple assay and that it does offer us the opportunity to study protein-protein interactions in a living cell (16Prichard L. Deloulme J.C. Storm D.R. J. Biol. Chem. 1999; 274: 7689-7694Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).The recent resolution of the structure of CaM associated with the SK-binding domain has shown that the binding domain is composed of two α-helices connected by a short loop (23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar). The high probability that the two KCNQ regions contain α-helices suggests that a similar conformation to the SK2 CaM-binding domain may also arise in KCNQ. However, some differences are evident. Although in SK channels the connecting loop is only 5 aa long, in KCNQ channels this varies from ∼100 to ∼150 aa. Secondly, the C lobe of CaM (EF hands 3 and 4) is sufficient for binding to SK channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar), whereas the complete CaM molecule is necessary for binding to KCNQ2 or KCNQ3 channels. In essence, our data suggests that the C-terminal domain of KCNQ channels folds in such a way that helix A and helix B form a compact structure that can be engulfed between the N- and C-terminal lobes of apo-CaM (Fig. 8).What is the role of CaM in KCNQ function? The preassociation of apo-CaM to a target protein whose activity may be regulated by CaM generally ensures a rapid and selective response to local elevations in Ca2+ (8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar, 27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Indeed, the suppression of the M current by bradykinin in rat sympathetic neurons is mediated by this cation (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar), and apo-CaM has been shown to modulate the Ca2+-dependent gating of many channels (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar,35Levitan I.B. Neuron. 1999; 22: 645-648Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The Ca2+-dependent modulation of M channels can also be observed in excised patches. Under these conditions, signaling pathways such as phosphorylation are not supported, and because the effects observed are fully reversible, it becomes very unlikely that the modulation of I Mvia Ca2+ is due to dephosphorylation of the M channel or of an associated protein. Furthermore, the absence of a "signature" flickering behavior and the tendency to "desensitize" in inside-out patches (i.e. the effect is transient) suggests that the activity of Ca2+ does not involve the direct blockage of the internal mouth of the channel (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These observations indicate that the mediator exists in limited amounts in excised patches and that it is washed out after the application of Ca2+. These properties suggest the involvement of a Ca2+ sensor such as CaM, which, as we have shown, interacts with the C-terminal intracellular region of KCNQ channels. In this respect, it is interesting to reflect on the fact that helix A is adjacent to the end of S6, the last transmembrane domain (Fig. 8). In other channels with a similar six transmembrane architecture, such as SK Ca2+-activated K+ channels and cyclic nucleotide-gated channels, gating is modulated by a module that attaches to the end of S6 (23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar, 36Johnson J.P.J. Zagotta W.N. Nature. 2001; 412: 917-921Crossref PubMed Scopus (93) Google Scholar). However, we should also bear in mind that the binding of CaM may also be important in other processes such as assembly or trafficking (37Gao T. Bunemann M. Gerhardstein B.L. Ma H. Hosey M.M. J. Biol. Chem. 2000; 275: 25436-25444Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 38Joiner W.J. Khanna R. Schlichter L.C. Kaczmarek L.K. J. Biol. Chem. 2001; 31: 699-711Google Scholar). The functional analysis of mutants unable to bind CaM should help to unveil the role of CaM in KCNQ channel function. The KCNQ transmembrane proteins are members of a family of voltage-dependent potassium selective channels that are involved in the control of cellular excitability. Remarkably, mutations in four of the five known members of this family have been associated with different hereditary human disorders. While mutations in the KCNQ1 subunit (KvQT1) lead to arrhythmia in the human long QT syndrome, mutations in KCNQ2 or KCNQ3 are associated with a benign form of epilepsy. It has also been shown that KCNQ4 is mutated in a dominant form of progressive hearing loss (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar). With regards to the normal physiology of this protein family, the KCNQ2 and KCNQ3 subunits have been shown to form M-type potassium channels whose expression is restricted to neuronal tissue (2Wang H.S. Pan Z. Shi W. Brown B.S. Wymore R.S. Cohen I.S. Dixon J.E. McKinnon D. Science. 1998; 282: 1890-1893Crossref PubMed Scopus (1015) Google Scholar). Moreover, in some brain areas and neuronal tissues, KCNQ4 and KCNQ5 also contribute to the formation of M channels, suggesting that the different combinations of KCNQ subunits may be in part responsible for the diversity of M channel properties (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar). The M current (I M) is a subthreshold noninactivating voltage-dependent potassium current that is found in many neuronal cell types. The M current controls membrane excitability, and it has been shown to be modulated by a variety of intracellular signals that in turn dramatically affect the firing rate of neurons. Among those intracellular signals, Ca2+ has been shown to mediate the inhibition of I M by B2bradykinin receptors in sympathetic neurons (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar). Indeed, intracellular Ca2+ can suppress the activity of M channels under conditions that do not support enzymatic activities such as phosphorylation (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This phenomenon suggests that an intermediary might be involved in this Ca2+-dependent modulation. In a search for candidates that might mediate the effects of Ca2+ in modulating I M, we screened a human brain cDNA library using the yeast two-hybrid system. We found that calmodulin (CaM)1bound to the C-terminal region of KCNQ channels. CaM is a small Ca2+-binding protein that acts as a ubiquitous intracellular Ca2+ sensor in the regulation of a growing diverse array of ion channels (5Saimi Y. Kung C. Annu. Rev. Physiol. 2002; 64: 289-311Crossref PubMed Scopus (290) Google Scholar). Efforts to define common characteristics of CaM binding have indicated that it associates with short α-helical sequences within its targets (6O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 7Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar, 8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar). However, it appears that the interaction of CaM with KCNQ channels does not conform to this simple model. Rather, our data suggest that the CaM-binding site in KCNQ channels is formed by two α-helices that are separated by a stretch of ∼130 amino acids. We hypothesize that those two helices come into close proximity in the tertiary structure, facilitating CaM binding. DISCUSSIONWe have demonstrated here that CaM binds to the intracellular C-terminal domain of neuronal KCNQ2, KCNQ3, and KCNQ5 transmembrane channels. Two-hybrid experiments suggest that CaM also binds to KCNQ1 and KCNQ4, but more experiments are necessary to unequivocally confirm this interaction. The voltage-dependent channels to which members of this family of proteins contribute have been implicated in a variety of physiological processes and pathologies. As a result, the modulation of the activity of these channels through intracellular signaling is important in maintaining the physiological homeostasis of nervous tissue (30Marrion N.V. Annu. Rev. Physiol. 1997; 59: 483-504Crossref PubMed Scopus (294) Google Scholar). An example of this can be seen in the Ca2+-dependent regulation of M channels (made up of KCNQ subunits) that influences the firing rate of the sympathetic cells in which they are expressed. Our results indicate that through its association with these proteins, CaM may mediate the Ca2+-dependent modulation of channels that include KCNQ subunits.In addition to demonstrating here that CaM associates with members of the KCNQ family, we have also defined its binding site. The binding site identified in this study is unusual in that it is composed of two discontinuous regions, helix A and helix B. Helix A contains an IQ-like binding motif, a motif (IQXXXRXXXXR) that mediates apo-CaM binding in a variety of proteins (31Bahler M. Rhoads A. FEBS Lett. 2002; 513: 107-113Crossref PubMed Scopus (361) Google Scholar) and that contains positively charged residues at positions 6 and 11. When compared with other IQ motifs, in the helix A of the KCNQ channels the second Arg is replaced by a negatively charged or neutral amino acid. As a result, this domain resembles the second "incomplete" IQ motif on myosin II, the region to which the regulatory myosin light chain (structurally similar to CaM) binds. A model of apo-CaM binding derived from this and other light chain structures bound to myosin IQ motifs reveals that the initial portion of this motif (IQXXXR) is the most critical part. Moreover, it is this region that is specifically recognized by the loop between EF hands 3 and 4 and that determines a semi-open lobe conformation (32Houdusse A. Silver M. Cohen C. Structure. 1996; 4: 1475-1490Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In accordance with this model, the interaction of KCNQ2 with CaM is destabilized when point mutations are introduced in this first part of the IQ motif.On many occasions, the C lobe of CaM has been shown to be that which interacts with peptides, as also occurs with CaM-like proteins that associate with peptides. Moreover, the bound peptide essentially occupies the same position relative to the C-terminal EF hand domain (33Atkinson R.A. Joseph C. Kelly G. Muskett F.W. Frenkiel T.A. Nietlispach D. Pastore A. Nat. Struct. Biol. 2001; 8: 853-857Crossref PubMed Scopus (73) Google Scholar). The finding that the C lobe is sufficient to bind to SK channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar), P/Q Ca2+ channels, and neurogranin supports this finding. However, in contrast, it appears that to bind to KCNQ2 or KCNQ3 channels, both the C and N lobes are required.Surprisingly, we found that when using the full-length C-terminal region of SK2 as bait, point mutations that abolish Ca2+binding to EF hands 3 or 4 also abrogate the interaction with CaM. There is, however, ample biochemical, functional, and structural evidence to indicate that Ca2+ is not bound to EF hands 3 or 4 when CaM interacts with SK2. In addition, it has been shown that the Ca2+-free C-terminal lobe is that which mediates the binding of CaM to the SK2 CaM-binding domain (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar). Our results indicate that mutating the first aspartate to alanine in the EF hands that directly mediate the interaction of apo-CaM with the target causes a reduction in the binding strength. By analogy with the SK2 CaM-binding domain, the observation that the interaction of CaM with KCNQ2 or KCNQ3 does not tolerate point mutations at EF hand 1 and (to a lesser degree) 3 suggests that binding to the KCNQ-BD is mainly mediated by the apo-EF hands 1 and 3. Interestingly, EF hand 1 is most similar to EF hand 3 (8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar), suggesting that both play a similar role in stabilizing the target complex.The difficulties in identifying apo-CaM interactions have been highlighted by Erickson et al. (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). To overcome this problem, a very elegant technique has been developed, three-cube fluorescence resonance energy transfer, that demonstrates the preassociation of CaM with calcium channels in living cells (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). The yeast two-hybrid system is another viable alternative for approaching this problem. Our results provide further evidence that the two-hybrid assay is capable of detecting interactions between the Ca2+-free form of CaM and target proteins such as the SK2 K+ channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar). It should be born in mind that a limitation of the two-hybrid system is that the transmembrane segments of target proteins must be eliminated to allow targeting of the bait to the nucleus (34Niethammer M. Sheng M. Methods Enzymol. 1998; 293: 104-122Crossref PubMed Scopus (26) Google Scholar). However, the main advantage is that this is a relatively simple assay and that it does offer us the opportunity to study protein-protein interactions in a living cell (16Prichard L. Deloulme J.C. Storm D.R. J. Biol. Chem. 1999; 274: 7689-7694Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).The recent resolution of the structure of CaM associated with the SK-binding domain has shown that the binding domain is composed of two α-helices connected by a short loop (23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar). The high probability that the two KCNQ regions contain α-helices suggests that a similar conformation to the SK2 CaM-binding domain may also arise in KCNQ. However, some differences are evident. Although in SK channels the connecting loop is only 5 aa long, in KCNQ channels this varies from ∼100 to ∼150 aa. Secondly, the C lobe of CaM (EF hands 3 and 4) is sufficient for binding to SK channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar), whereas the complete CaM molecule is necessary for binding to KCNQ2 or KCNQ3 channels. In essence, our data suggests that the C-terminal domain of KCNQ channels folds in such a way that helix A and helix B form a compact structure that can be engulfed between the N- and C-terminal lobes of apo-CaM (Fig. 8).What is the role of CaM in KCNQ function? The preassociation of apo-CaM to a target protein whose activity may be regulated by CaM generally ensures a rapid and selective response to local elevations in Ca2+ (8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar, 27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Indeed, the suppression of the M current by bradykinin in rat sympathetic neurons is mediated by this cation (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar), and apo-CaM has been shown to modulate the Ca2+-dependent gating of many channels (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar,35Levitan I.B. Neuron. 1999; 22: 645-648Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The Ca2+-dependent modulation of M channels can also be observed in excised patches. Under these conditions, signaling pathways such as phosphorylation are not supported, and because the effects observed are fully reversible, it becomes very unlikely that the modulation of I Mvia Ca2+ is due to dephosphorylation of the M channel or of an associated protein. Furthermore, the absence of a "signature" flickering behavior and the tendency to "desensitize" in inside-out patches (i.e. the effect is transient) suggests that the activity of Ca2+ does not involve the direct blockage of the internal mouth of the channel (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These observations indicate that the mediator exists in limited amounts in excised patches and that it is washed out after the application of Ca2+. These properties suggest the involvement of a Ca2+ sensor such as CaM, which, as we have shown, interacts with the C-terminal intracellular region of KCNQ channels. In this respect, it is interesting to reflect on the fact that helix A is adjacent to the end of S6, the last transmembrane domain (Fig. 8). In other channels with a similar six transmembrane architecture, such as SK Ca2+-activated K+ channels and cyclic nucleotide-gated channels, gating is modulated by a module that attaches to the end of S6 (23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar, 36Johnson J.P.J. Zagotta W.N. Nature. 2001; 412: 917-921Crossref PubMed Scopus (93) Google Scholar). However, we should also bear in mind that the binding of CaM may also be important in other processes such as assembly or trafficking (37Gao T. Bunemann M. Gerhardstein B.L. Ma H. Hosey M.M. J. Biol. Chem. 2000; 275: 25436-25444Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 38Joiner W.J. Khanna R. Schlichter L.C. Kaczmarek L.K. J. Biol. Chem. 2001; 31: 699-711Google Scholar). The functional analysis of mutants unable to bind CaM should help to unveil the role of CaM in KCNQ channel function. We have demonstrated here that CaM binds to the intracellular C-terminal domain of neuronal KCNQ2, KCNQ3, and KCNQ5 transmembrane channels. Two-hybrid experiments suggest that CaM also binds to KCNQ1 and KCNQ4, but more experiments are necessary to unequivocally confirm this interaction. The voltage-dependent channels to which members of this family of proteins contribute have been implicated in a variety of physiological processes and pathologies. As a result, the modulation of the activity of these channels through intracellular signaling is important in maintaining the physiological homeostasis of nervous tissue (30Marrion N.V. Annu. Rev. Physiol. 1997; 59: 483-504Crossref PubMed Scopus (294) Google Scholar). An example of this can be seen in the Ca2+-dependent regulation of M channels (made up of KCNQ subunits) that influences the firing rate of the sympathetic cells in which they are expressed. Our results indicate that through its association with these proteins, CaM may mediate the Ca2+-dependent modulation of channels that include KCNQ subunits. In addition to demonstrating here that CaM associates with members of the KCNQ family, we have also defined its binding site. The binding site identified in this study is unusual in that it is composed of two discontinuous regions, helix A and helix B. Helix A contains an IQ-like binding motif, a motif (IQXXXRXXXXR) that mediates apo-CaM binding in a variety of proteins (31Bahler M. Rhoads A. FEBS Lett. 2002; 513: 107-113Crossref PubMed Scopus (361) Google Scholar) and that contains positively charged residues at positions 6 and 11. When compared with other IQ motifs, in the helix A of the KCNQ channels the second Arg is replaced by a negatively charged or neutral amino acid. As a result, this domain resembles the second "incomplete" IQ motif on myosin II, the region to which the regulatory myosin light chain (structurally similar to CaM) binds. A model of apo-CaM binding derived from this and other light chain structures bound to myosin IQ motifs reveals that the initial portion of this motif (IQXXXR) is the most critical part. Moreover, it is this region that is specifically recognized by the loop between EF hands 3 and 4 and that determines a semi-open lobe conformation (32Houdusse A. Silver M. Cohen C. Structure. 1996; 4: 1475-1490Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In accordance with this model, the interaction of KCNQ2 with CaM is destabilized when point mutations are introduced in this first part of the IQ motif. On many occasions, the C lobe of CaM has been shown to be that which interacts with peptides, as also occurs with CaM-like proteins that associate with peptides. Moreover, the bound peptide essentially occupies the same position relative to the C-terminal EF hand domain (33Atkinson R.A. Joseph C. Kelly G. Muskett F.W. Frenkiel T.A. Nietlispach D. Pastore A. Nat. Struct. Biol. 2001; 8: 853-857Crossref PubMed Scopus (73) Google Scholar). The finding that the C lobe is sufficient to bind to SK channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar), P/Q Ca2+ channels, and neurogranin supports this finding. However, in contrast, it appears that to bind to KCNQ2 or KCNQ3 channels, both the C and N lobes are required. Surprisingly, we found that when using the full-length C-terminal region of SK2 as bait, point mutations that abolish Ca2+binding to EF hands 3 or 4 also abrogate the interaction with CaM. There is, however, ample biochemical, functional, and structural evidence to indicate that Ca2+ is not bound to EF hands 3 or 4 when CaM interacts with SK2. In addition, it has been shown that the Ca2+-free C-terminal lobe is that which mediates the binding of CaM to the SK2 CaM-binding domain (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar). Our results indicate that mutating the first aspartate to alanine in the EF hands that directly mediate the interaction of apo-CaM with the target causes a reduction in the binding strength. By analogy with the SK2 CaM-binding domain, the observation that the interaction of CaM with KCNQ2 or KCNQ3 does not tolerate point mutations at EF hand 1 and (to a lesser degree) 3 suggests that binding to the KCNQ-BD is mainly mediated by the apo-EF hands 1 and 3. Interestingly, EF hand 1 is most similar to EF hand 3 (8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar), suggesting that both play a similar role in stabilizing the target complex. The difficulties in identifying apo-CaM interactions have been highlighted by Erickson et al. (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). To overcome this problem, a very elegant technique has been developed, three-cube fluorescence resonance energy transfer, that demonstrates the preassociation of CaM with calcium channels in living cells (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). The yeast two-hybrid system is another viable alternative for approaching this problem. Our results provide further evidence that the two-hybrid assay is capable of detecting interactions between the Ca2+-free form of CaM and target proteins such as the SK2 K+ channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar). It should be born in mind that a limitation of the two-hybrid system is that the transmembrane segments of target proteins must be eliminated to allow targeting of the bait to the nucleus (34Niethammer M. Sheng M. Methods Enzymol. 1998; 293: 104-122Crossref PubMed Scopus (26) Google Scholar). However, the main advantage is that this is a relatively simple assay and that it does offer us the opportunity to study protein-protein interactions in a living cell (16Prichard L. Deloulme J.C. Storm D.R. J. Biol. Chem. 1999; 274: 7689-7694Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The recent resolution of the structure of CaM associated with the SK-binding domain has shown that the binding domain is composed of two α-helices connected by a short loop (23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar). The high probability that the two KCNQ regions contain α-helices suggests that a similar conformation to the SK2 CaM-binding domain may also arise in KCNQ. However, some differences are evident. Although in SK channels the connecting loop is only 5 aa long, in KCNQ channels this varies from ∼100 to ∼150 aa. Secondly, the C lobe of CaM (EF hands 3 and 4) is sufficient for binding to SK channels (21Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar), whereas the complete CaM molecule is necessary for binding to KCNQ2 or KCNQ3 channels. In essence, our data suggests that the C-terminal domain of KCNQ channels folds in such a way that helix A and helix B form a compact structure that can be engulfed between the N- and C-terminal lobes of apo-CaM (Fig. 8). What is the role of CaM in KCNQ function? The preassociation of apo-CaM to a target protein whose activity may be regulated by CaM generally ensures a rapid and selective response to local elevations in Ca2+ (8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar, 27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Indeed, the suppression of the M current by bradykinin in rat sympathetic neurons is mediated by this cation (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar), and apo-CaM has been shown to modulate the Ca2+-dependent gating of many channels (27Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar,35Levitan I.B. Neuron. 1999; 22: 645-648Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The Ca2+-dependent modulation of M channels can also be observed in excised patches. Under these conditions, signaling pathways such as phosphorylation are not supported, and because the effects observed are fully reversible, it becomes very unlikely that the modulation of I Mvia Ca2+ is due to dephosphorylation of the M channel or of an associated protein. Furthermore, the absence of a "signature" flickering behavior and the tendency to "desensitize" in inside-out patches (i.e. the effect is transient) suggests that the activity of Ca2+ does not involve the direct blockage of the internal mouth of the channel (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These observations indicate that the mediator exists in limited amounts in excised patches and that it is washed out after the application of Ca2+. These properties suggest the involvement of a Ca2+ sensor such as CaM, which, as we have shown, interacts with the C-terminal intracellular region of KCNQ channels. In this respect, it is interesting to reflect on the fact that helix A is adjacent to the end of S6, the last transmembrane domain (Fig. 8). In other channels with a similar six transmembrane architecture, such as SK Ca2+-activated K+ channels and cyclic nucleotide-gated channels, gating is modulated by a module that attaches to the end of S6 (23Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (490) Google Scholar, 36Johnson J.P.J. Zagotta W.N. Nature. 2001; 412: 917-921Crossref PubMed Scopus (93) Google Scholar). However, we should also bear in mind that the binding of CaM may also be important in other processes such as assembly or trafficking (37Gao T. Bunemann M. Gerhardstein B.L. Ma H. Hosey M.M. J. Biol. Chem. 2000; 275: 25436-25444Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 38Joiner W.J. Khanna R. Schlichter L.C. Kaczmarek L.K. J. Biol. Chem. 2001; 31: 699-711Google Scholar). The functional analysis of mutants unable to bind CaM should help to unveil the role of CaM in KCNQ channel function. We are very grateful to Drs. J. Maylie (Oregon Health Sciences University), T. Jentsch (Zentrum für Molekulare Neurobiologie Hamburg, Hamburg, Germany), L. M. Pardo and W. Stühmer (Max Planck Institute, Göttingen, Germany), A. Villalobo and J. Bernal (Instituto de Investigaciones Biologicas-Consejo Superior de Investigaciones Cientificas, Madrid, Spain), W. A. Catterall (University of Washington), B. J. Jensen (NeuroSearch, Ballerup, Denmark), V. I. Teichberg (Weizmann Institute of Science, Rehovot, Israel), J. T. Stull (University of Texas Southwestern Medical Center, Dallas, TX), and S. Pons (I Cajal, Madrid, Spain) for providing us with cDNAs and other materials used in this study. We thank Dr. Paula Bosch and Dr. Fernando Diaz for providing equipment and help in the fluorimetric assays. We acknowledge the help of Dr. Fernando Moro and Dr. M. Paz Regalado in some parts of the project, Dr. Mark Sefton for critical comments on the manuscript and editorial help, and Carmen Page and Uyen Le for technical assistance.
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